Compositions and methods for use of recombinant t cell receptors for direct recognition of tumor antigen

ABSTRACT

Provided are compositions and methods for prophylaxis and/or therapy of a variety of cancers which express a NY-ESO-1 antigen. Included are recombinant T cell receptors (TCRs), polynucleotides encoding them, expression vectors that include the polynucleotides, and cells into which the polynucleotides have been introduced to produce modified cells, including CD4 +  T cells, CD8 +  T cells, natural killer T cells, γδ T cells, and progenitor cells, such as haematopoietic stem cells. The modified cells are capable of direct recognition of a cancer cell expressing a NY-ESO-1 antigen by human leukocyte antigen (HLA) class II-restricted binding of the TCR to the NY-ESO-1 antigen expressed by the cancer cell without presentation of the antigen by antigen presenting cells. In embodiments, the NY-ESO-1 antigen is displayed by the tumor cells. Also included is a method for prophylaxis and/or therapy of cancer by administering modified cells that express a recombinant TCR. Methods for making expression vectors and/or cells which express a recombinant TCR and identifying TCRs to make the expression vectors are also included.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. application No. 61/778,673,filed Mar. 13, 2013, the disclosure of which is incorporated herein byreference.

FIELD

The present disclosure relates generally to immunotherapy and morespecifically to recombinant T cell receptors that can impart directtumor recognition capability to T cells.

BACKGROUND OF THE INVENTION

Tumor antigen-specific CD4⁺ helper T cells play critical roles in theinduction and maintenance of anti-tumor immune responses by providing“CD4-help”. Activation of CD4⁺ T cells at the local tumor sites isbelieved to help overcome multiple immuno-suppression mechanisms andpromote tumor eradication by the immune system. However, because of thefrequent lack of functional antigen-presenting cells at the local tumorsites, activation of the CD4⁺ T cells and therefore the provision ofCD4-help at the local tumor site is severely limited. There isaccordingly an ongoing and unmet need to provide new compositions andmethods such that activation of CD4⁺ T cells and therefore provision ofCD4-help can be achieved.

SUMMARY

The present disclosure provides compositions and methods for prophylaxisand/or therapy of a variety of cancers. In general, the cancers arethose which express the well-known the NY-ESO-1 antigen. In embodiments,the disclosure includes recombinant T cell receptors (TCRs),polynucleotides encoding them, expression vectors comprising thepolynucleotides, cells into which the polynucleotides have beenintroduced, including but not necessarily limited CD4⁺ T cells, CD8⁺ Tcells, natural killer T cells, γδ T cells, and progenitor cells, such ashaematopoietic stem cells. In embodiments, the cells into which thepolynucleotides are introduced are lymphoid progenitor cells, immaturethymocytes (double-negative CD4−CD8−) cells, or double-positivethymocytes (CD4+CD8+). In embodiments, the progenitor cells comprisemarkers, such as CD34, CD117 (c-kit) and CD90 (Thy-1).

In one aspect the disclosure includes a modified human T cell comprisinga recombinant polynucleotide encoding a TCR, wherein the T cell iscapable of direct recognition of a cancer cell expressing a NY-ESO-1antigen, wherein the direct recognition of the cancer cell compriseshuman leukocyte antigen (HLA) class II-restricted binding of the TCR tothe NY-ESO-1 antigen expressed by the cancer cell. In particularembodiments, the TCR encoded by the polynucleotide and expressed by thecell has a TCR alpha chain having the sequence of SEQ ID NO:3 and a TCRbeta chain having the sequence of SEQ ID NO:4, or a TCR alpha chainhaving the sequence of SEQ ID NO:7 and a TCR beta chain having thesequence of SEQ ID NO:8, or a TCR alpha chain having the sequence of SEQID NO:11 and a TCR beta chain having the sequence of SEQ ID NO:12. Allcombination of such alpha and beta chains are included in thedisclosure. In an embodiment, the modified cell of claim 1, wherein thesequence encoding the alpha chain and/or the beta chain does notcomprise introns. In embodiments, the TCRs of this disclosure includeamino acid sequences that are 95%, 96%, 97%, 98%, or 99% amino acidsequence identify across the length of the amino acid sequencesdisclosed herein.

In another aspect the disclosure includes a method for prophylaxisand/or therapy of an individual diagnosed with, suspected of having orat risk for developing or recurrence of a cancer, wherein the cancercomprises cancer cells which express NY-ESO-1 antigen. This approachcomprises administering to the individual modified human T cellscomprising a recombinant polynucleotide encoding a TCR, wherein the Tcells are capable of direct recognition of the cancer cells expressingthe NY-ESO-1 antigen, and wherein the direct recognition of the cancercells comprises HLA class II-restricted binding of the TCR to theNY-ESO-1 antigen expressed by the cancer cells. In embodiments, thecells comprising the recombinant TCR are human CD4⁺ T cells. Inembodiments, the cells comprising the recombinant TCR that isadministered to the individual are allogeneic, syngeneic, or autologouscells. Thus, in one embodiment, the cells are obtained from a firstindividual, modified, and administered to a second individual who is inneed thereof. In another embodiment, the cells are removed from theindividual prior, modified to express the recombinant TCR, andadministered back to the same individual.

In embodiments, the cancer that expresses the NY-ESO-1 antigen isselected from bladder cancer cells, brain cancer cells, breast cancercells, gastric cancer cells, esophageal cancer cells, head and neckcancer cells, hepatobiliary cancer cells, kidney cancer cells, ovarycancer cells, non-small cell lung cancer cells, myeloma, prostate cancercells, sarcoma cells, testicular cancer cells, melanoma cells, andcombinations thereof.

In another aspect the disclosure includes one or more expressionvectors. The expression vector(s) encode a TCR that is capable ofimparting to a cell which expresses it the capability to directly acancer cell expressing a NY-ESO-1 antigen, wherein the directrecognition of the cancer cell comprises HLA class II-restricted bindingof the TCR to the NY-ESO-1 antigen expressed by the cancer cell.

In another approach, methods for making expression vectors and/or cellswhich express a recombinant TCR. The method involves obtaining aplurality of T cells from an individual, identifying T cells that arecapable of direct recognition of a cancer cell displaying a NY-ESO-1antigen in an HLA class II-restricted manner without antigen presentingcells presenting the NY-ESO-1 antigen to the T cells, determining thesequence of the alpha chain of the TCR and the sequence of the betachain of the TCR, and introducing into an expression vector apolynucleotide sequence encoding the alpha chain of the TCR and the betachain of the TCR. In an embodiment, this method comprises introducingthe expression vector into a cell such that the TCR is expressed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. (A) Direct recognition of cancer cells by JM CD4⁺ T cell clone.Interferon (IFN)-γ and CD107 expression of NY-ESO-1₁₅₇₋₁₇₀peptide-specific tumor-recognizing CD4⁺ T cell clone (Clone: JM)(TR-CD4) and non-tumor-recognizing CD4⁺ T cell clone (NTR-CD4) aftercoculture with NY-ESO-1-expressing SK-MEL-37 (SK37) andNY-ESO-1-negative SK-MEL-29 (SK29) with or without pulsing with thecognate NY-ESO-1₁₅₇₋₁₇₀ (ESO-1₁₅₇₋₁₇₀) peptide was investigated byintracellular cytokine staining. (B) Differences in intracellular andextracellular NY-ESO-1 recognition by NY-ESO-1-specific CD4⁺ and CD8⁺ Tcell clones. NY-ESO-1-negative SK-MEL-29 was unpulsed (Unpulsed) orpulsed with NY-ESO-1₁₅₇₋₁₇₀ peptide (Peptide) or recombinant NY-ESO-1protein (Protein), or was infected with adenovirus vector which induceintracellular NY-ESO-1 expression. Recognition by TR-CD4, NTR-CD4 andNY-ESO-1-specific CD8⁺ T cell clone was evaluated by IFN-γ ELISPOTassay.

FIG. 2. (A) TR-CD4 (Clone: JM) were co-cultured with SK-MEL-37. Culturesupernatant was harvested after 1-4 days of culture. The levels of theindicated cytokines and lytic molecules in the supernatant were measuredby ELISA. (B) TR-CD4 and NTR-CD4 was co-cultured with SK-MEL-37 andexpression of the early apoptosis marker, Annexin-V, on SK-MEL-37 (SK37)was measured by flow-cytometry.

FIG. 3. NY-ESO-1-specific CD8⁺ T cell clone (ESO-CD8) was co-culturedwith SK-MEL-37 at 1:2 ratio in the presence or absence of the indicatedratios of TR-CD4 (Clone: JM). Cytotoxic activity by ESO-CD8 on SK-MEL-37was evaluated by CFSE-based cytotoxicity assay.

FIG. 4. (A) NY-ESO-1-specific CD8⁺ T cell clone (ESO-CD8) was stimulatedwith or without SK-MEL-37 (SK37) in the presence or absence of TR-CD4(clone: JM). After 4 days, the number of CD8⁺ T cells were enumerated bytrypan blue exclusion assay combined with CD8 staining byflow-cytometry. (B) ESO-CD8 was stimulated with SK37 in the presence orabsence of TR-CD4. Before (day 0) and after (day 1 and day 2)stimulation, expression of activation markers (CD25, CD69 and CD122) orcentral T cell differentiation markers (CD62L, CCR7 and CD127) onESO-CD8 was measured by flow-cytometry.

FIG. 5. (A) SK-MEL-37 was inoculated in SCID mice (6 mice/group) with orwithout tumor-recognizing CD4⁺ T cell clone (JM: TR-CD4),non-tumor-recognizing CD4⁺ T cell clone (NTR-CD4), and/orNY-ESO-1-specific CD8⁺ T cell clone (ESO-CD8). Tumor growth was measuredevery 2-3 day. (B) Tumor was excised and weighted at day 45 afterinoculation.

FIG. 6. (A) Retrovirus vector used in the experiments. LTR:long-terminal repeat; ψ: packaging signal; MCS: multiple cloning site;IRES: internal ribosome entry site; eGFP: enhanced green fluorescentprotein. (B) TCR expressing cassette. (I) TCR β and α chain-coding cDNAsequences are connected by a GSG (Gly-Ser-Gly) linker and a P2Aribosomal skipping sequence. (II) TCR β and α chain-coding cDNAsequences are connected by a furin protease recognition site (RAKR(Arg-Ala-Lys-Arg)), a SGSG (Ser-Gly-Ser-Gly) linker, V5 epitope, and aP2A ribosomal skipping sequence.

FIG. 7. Polyclonally activated PBMC were transduced with retroviralvector (A: JM-TCR; B: SB95-TCR). They were cocultured withpeptide-pulsed (Pulsed) or unpulsed (Unpulsed) HLA-DRB1*01+DPB1*04+cells for 20 hours. IFN-γ level in the supernatant was measured byELISA. NY-ESO-1₁₅₇₋₁₇₀ and NY-ESO-1₉₁₋₁₁₀ peptides were used as thecognate peptides for JM-TCR and SB95-TCR, respectively.

DESCRIPTION OF THE INVENTION

The present disclosure relates to immune cells, including but notnecessarily limited to T cells, that have been engineered to be capableof direct recognition of tumor antigen and MHC class II-expressingcancer cells. In embodiments, the immune cells are CD4⁺ T cells, CD8⁺ Tcells, natural killer T cells, γδ T cells, or their progenitor cellssuch hematopoietic stem/progenitor cells. In embodiments, thehematopoietic/progenitor cells are characterized by one or more markersselected from CD34, CD117 (c-kit) and CD90 (Thy-1).

It is well known that CD4⁺ T cells typically recognize peptide fragmentspresented on MHC class II (HLA class II in humans) by antigen presentingcells, such as macrophages and dendritic cells. In addition toantigen-presenting cells, many human cancer cells are also known toexpress MHC class II constitutively or in an IFN-γ-inducible manner, butthe role of MHC class II expression on human cancer cells remainslargely unknown.

We have now discovered that there are two distinct types of tumorantigen-specific CD4⁺ T cells. One type of tumor antigen-specific CD4⁺ Tcells is referred to herein as tumor-recognizing CD4⁺ T cells (TR-CD4).This type of CD4⁺ T cell directly recognizes MHC (HLA in humans) classII-expressing cancer cells in antigen-specific and MHC classII-restricted manner. In contrast, another type of previously known,antigen-specific CD4⁺ T cells is referred to herein asnon-tumor-recognizing CD4⁺ T cells (NTR-CD4). This type of T cell onlyrecognizes exogenous tumor antigen peptides after processing byantigen-presenting cells. FIGS. 1A and 1B depict data demonstratingthese distinct functions and reveal direct recognition of cancer cellsby TR-CD4.

Because of their different abilities in direct recognition of cancercells, these two types of CD4⁺ T cells (TR-CD4 and NTR-CD4) are believedto play different roles at the local tumor site. Without intending to beconstrained by any particular theory, it is believed that TR-CD4 cellsprovide CD4-help by direct recognition of cancer cells. The presentinvention takes advantage of this function to provide TCR polypeptidesand recombinant polynucleotides encoding them for use in novelprophylactic and/or therapeutic treatment modalities and compositions.By engineering T cells to express the TCRs further described herein, wecan endow any CD4⁺ cell with the capability to directly recognize tumorantigen-expressing cancer cells, without requiring presentation of theantigen by an antigen-presenting cell. Thus, the present inventionincludes compositions and methods that are useful for creating and usingTR-CD4 cells for improved care of cancer patients.

Previous attempts at making and using recombinant TCRs have been made.For example, U.S. Pat. No. 8,008,438 (the '438 patent) disclosesrecombinant TCRs which bind to the peptide sequence SLLMWITQC from theNY-ESO-1 protein (NY-ESO-1:157-165). However, and importantly, thedisclosure in the '438 patent pertains to classic CD8⁺ TCRs, which onlyrecognize the NY-ESO-1:157-165 peptide in the context of the HLA-A*0201class I restriction element. This constitutes a significantdissimilarity from the present invention because, as described above,the recombinant TCRs of the present invention are class II restricted.Moreover, and as also described above, unlike canonical class IIrestriction, cells engineered to express a recombinant TCR of theinvention surprisingly do not require the assistance of antigenpresenting cells to recognize the antigens to which they are specific.Instead, they can recognize the antigens as they exist in vivo as apeptide displayed by the tumor cells. Further, the TCRs of the presentinvention recognize peptides by those disclosed in the '438 patent.Accordingly, the present invention is a significant and unexpecteddeparture from the prior art. In an embodiment, a TR-CD4 is a CD4+ cellthat exhibits cytokine secretion (such as IFN-gamma production) when theTR-CD4 is directly exposed to cells which express an antigen for whichthe TCR is specific in an HLA-II context. The ability to confercapability for direct recognition of NY-ESO-1-expressing tumors by CD4+T cells by introducing a TCR from a naturally occurring cell having thiscapability was unexpected.

In one embodiment, the invention includes transforming any CD4⁺ T cellinto a TR-CD4 by introducing a polynucleotide encoding a recombinant TCRof the invention into polyclonally expanded CD4⁺ T cells and allowingexpression of the TCR polypeptide coding region(s) of thepolynucleotide.

In various embodiments, the present invention provides isolated and/orrecombinant polynucleotides encoding particular TCR polypeptides, cellsengineered to express the TCR polypeptides, pharmaceutical formulationscomprising cells which express the TCR polypeptides, and methods ofusing the pharmaceutical formulations to achieve a prophylactic and/ortherapeutic effect against cancer in a subject. In certain embodiments,the invention provides mixtures of cells expressing TCRs, or cellsexpressing more than one TCR described herein, that are specific fordistinct cancer antigens, thus presenting cell populations that can beconsidered polyvalent with respect to the TCRs. As used in thisdisclosure, a “recombinant TCR” means a TCR that is expressed from apolynucleotide that was introduced into the cell, meaning prior to theintroduction of the polynucleotide the TCR was not encoded by achromosomal sequence in the cell.

The TCRs provided by the invention are capable of recognizingNY-ESO-1;157-170 which is an antigen that consists of the amino acidsequence SLLMWITQCFLPVF, or are capable of recognizing NY-ESO-1;95-106,which is an antigen that consists of the amino acid sequencePFATPMEAELAR. As described above, in certain embodiments, the cellsprovided by the invention are engineered CD4⁺ T cells that are capableof recognizing these antigens via TCRs which interact with the antigenin association with HLA class II molecules, wherein the HLA class IImolecules and antigen are displayed by tumor cells.

The invention includes each and every polynucleotide sequence thatencodes one or more TCR polypeptides of the invention and disclosedherein, including DNA and RNA sequences, and including isolated and/orrecombinant polynucleotides comprising and/or consisting of suchsequences. The invention also includes cells which comprise therecombinant polynucleotides. The cells can be isolated cells, cellsgrown and/or expanded and/or maintained in culture, and can beprokaryotic or eukaryotic cells. Prokaryotic and eukaryotic cellcultures can be used, for example, to propagate or amplify the TCRexpression vectors of the invention. In embodiments, the cells cancomprise packaging plasmids, which, for example, provide some or all ofthe proteins used for transcription and packaging of an RNA copy of theexpression construct into recombinant viral particles, such aspseudoviral particles. In embodiments, the expression vectors aretransiently or stably introduced into cells. In embodiments, theexpression vectors are integrated into the chromosome of cells used fortheir production. In embodiments, polynucleotides encoding the TCRswhich are introduced into cells by way of an expression vector, such asa viral particle, are integrated into one or more chromosomes of thecells. Such cells can be used for propagation, or they can be cells thatare used for therapeutic and/or prophylactic approaches. The eukaryoticcells include CD4⁺ T cells, CD8⁺ T cells, natural killer T cells, γδ Tcells, and their progenitor cells into which a TCR expression constructof the invention has been introduced. The CD4⁺ T cells can be from anysource, including but not limited to a human subject who may or may notbe the eventual recipient of the CD4⁺ T cells once they have beenengineered to express a TCR according to the invention.

Expression vectors for use with embodiments of this disclosure can beany suitable expression vector. In embodiments, the expression vectorcomprises a modified viral polynucleotide, such as from an adenovirus, aherpesvirus, or a retrovirus, such as a lentiviral vector. Theexpression vector is not restricted to recombinant viruses and includesnon-viral vectors such as DNA plasmids and in vitro transcribed mRNA.

With respect to the polypeptides that are encoded by the polynucleotidesdescribed above, in certain aspects the invention provides functionalTCRs which comprises a TCR α and a TCR β chain, wherein the two chainsare present in a physical association with one another (e.g., in acomplex) and are non-covalently joined to one another, or wherein thetwo chains are distinct polypeptides but are covalently joined to oneanother, such as by a disulfide or other covalent linkage that is not apeptide bond. Other suitable linkages can comprise, for example,substituted or unsubstituted polyalkylene glycol, and combinations ofethylene glycol and propylene glycol in the form of, for example,copolymers. In other embodiments, two polypeptides that constitute theTCR α and a TCR β chain can both be included in a single polypeptide,such as a fusion protein. In certain embodiments, the fusion proteincomprises a TCR α chain amino acid sequence and a TCR β chain amino acidsequence that have been translated from the same open reading frame(ORF), or distinct ORFs, or an ORF that contain a signal that results innon-continuous translation. In one embodiment, the ORF comprises aP2A-mediated translation skipping site positioned between the TCR α andTCR β chain. Constructs for making P2A containing proteins (alsoreferred to as 2A Peptide-Linked multicistronic vectors) are known inthe art. (See, for example, Gene Transfer: Delivery and Expression ofDNA and RNA, A Laboratory Manual, (2007), Friedman et al., InternationalStandard Book Number (ISBN) 978-087969765-5. Briefly, 2A peptidesequences, when included between coding regions, allow forstoichiometric production of discrete protein products within a singlevector through a novel cleavage event that occurs in the 2A peptidesequence. 2A peptide sequences are generally short sequence comprising18-22 amino acids and can comprise distinct amino-terminal sequences.Thus, in one embodiment, a fusion protein of the invention includes aP2A amino acid sequence. In embodiments, a fusion protein of theinvention can comprise a linker sequence between the TCR α and TCR βchains. In certain embodiments, the linker sequence can comprise a GSG(Gly-Ser-Gly) linker or an SGSG (Ser-Gly-Ser-Gly) linker. In certainembodiments, the TCR α and TCR β chains are connected to one another byan amino acid sequence that comprises a furin protease recognition site,such as an RAKR (Arg-Ala-Lys-Arg) site.

In one embodiment, the expression construct that encodes the TCR canalso encode additional polynucleotides. The additional polynucleotidecan be such that it enables identification of TCR expressing cells, suchas by encoding a detectable marker, such as a fluorescent or luminescentprotein. The additional polynucleotide can be such that it encodes anelement that allows for selective elimination of TCR expressing cells,such as thymidine kinase gene. In embodiments the additionalpolynucleotides can be such that they facilitate inhibition ofexpression of endogenously encoded TCRs. In an embodiment, theexpression construct that encodes the TCR also encodes a polynucleotidewhich can facilitate RNAi-mediated down-regulation of one or moreendogenous TCRs For example, see Okamoto S, et al. (2009) CancerResearch, 69:9003-9011, and Okamoto S, et al. (2012). MolecularTherapy-Nucleic Acids, 1, e63. In an embodiment, the expressionconstruct that encodes the TCR can encode an shRNA or an siRNA targetedto an endogenously encoded TCR. In an alternative embodiment, a second,distinct expression construct that encodes the polynucleotide for use indownregulating endogenous TCR production can be used.

FIG. 6 provides representative configurations of TCR polypeptides of theinvention and polynucleotides/expression vectors encoding them. In oneembodiment, as outlined in FIG. 6, an amino acid sequence that isC-terminal to the TCR β chain protein is removed by furinprotease-mediated cleavage, resulting in functional TCR α and β chainproteins. It will be also recognized from FIG. 6 that the TCR chains canbe expressed from an expression construct such that the β chain isoriented N-terminally in relation to the α chain, and thus TCRs of theinvention can also comprise this chain orientation, or otherorientations. In alternative embodiments, the TCR α and β chain proteinscan be expressed from distinct expression vectors introduced into thesame cell.

In connection with the present invention, we have also made thefollowing discoveries: in certain instances, intracellular tumor antigenis loaded on HLA class II through recycling of the HLA class II intumors; direct tumor recognition by tumor-recognizing CD4⁺ T cells leadsto in vivo tumor growth inhibition; CD4⁺ T cells efficiently augmentCD8⁺ T cell cytotoxicity through direct tumor recognition; CD4⁺ T cellssupport proliferation, survival, and memory differentiation of cognateantigen-specific CD8⁺ T cells through direct tumor recognition withoutantigen presenting cells. It is expected that practicing the presentinvention in a clinical setting will also result in direct tumorrecognition by the engineered tumor-recognizing CD4⁺ T cells and lead toin vivo tumor growth inhibition in human subject, and will also resultin the efficient augmentation of CD8⁺ T cell cytotoxicity by theengineered CD4⁺ T cells, and that the engineered CD4⁺ T cells willsupport proliferation, survival, and memory differentiation of cognateantigen-specific CD8⁺ T cells in human subjects who receive CD4⁺ T cellsengineered according to the invention.

With respect to use of the engineered CD4⁺ T cells of the presentinvention, the method generally comprises administering an effectiveamount (typically 10¹⁰ cells by intravenous or intraperitonealinjections) of a composition comprising the CD4⁺ T cells to anindividual in need thereof. An individual in need thereof, in variousembodiments, is an individual who has or is suspected of having, or isat risk for developing a cancer which is characterized by malignantcells that express NY-ESO-I. As is well known in the art, NY-ESO-I isexpressed by a variety of cancer cells and tumor types. In particularand non-limiting examples, such cancers include cancers of the bladder,brain, breast, ovary, non-small cell lung cancer, myeloma, prostate,sarcoma and melanoma. Specific embodiments include but are not limitedto liposarcomas and intrahepatic cholagiocarcinoma. The individual mayhave early-stage or advanced forms of any of these cancers, or may be inremission from any of these cancers. In one embodiment, the individualto whom a composition of the invention is administered is at risk forrecurrence for any cancer type that expresses NY-ESO-1. In certainembodiments, the individual has or is suspected of having, or is at riskfor developing or recurrence of a tumor comprising cells which express aprotein comprising the amino acid sequences defined by NY-ESO-1:157-170and/or NY-ESO-1:95-106. In embodiments, the disclosure includesrecombinant TCRs that are specific for peptide fragments of NY-ESO-1that are between 15 and 24 amino acid residues long, wherein suchpeptides are presented in a complex with HLA-II. In embodiments, thedisclosure includes recombinant TCRs that are specific for peptides thatare in a complex with HLA-II, wherein the peptides comprise or consistof the amino acid sequences of NY-ESO-1:157-170 and/or NY-ESO-1:95-106.

The present disclosure includes recombinant TCRs, cells expressing them,and therapeutic/prophylactic methods that involve presentation ofNY-ESO-1 antigens in conjunction with any HLA-class II complex that willbe recognized by the TCRs. In embodiments, the HLA-II is selected fromHLA-DP, HLA-DQ, and HLA-DR. In embodiments, the NY-ESO-1 antigen isrecognized by the TCR in conjunction with HLA-DRB1*01 or HLA-DPB1*04.

We demonstrate in this invention that TR-CD4 we created produce multiplemolecules through direct recognition of cancer cells, which inducedapoptosis in cancer cells (FIGS. 2A and 2B) Importantly, TR-CD4 werefound to efficiently enhance the cytotoxic activity of tumorantigen-specific CD8⁺ T cells via direct recognition of cancer cells inthe absence of antigen-presenting cells (FIG. 3). Furthermore, CD8⁺ Tcells co-stimulated with TR-CD4 by cancer cells actively proliferatedand upregulated central memory T cell markers (FIGS. 4A and 4B).

TR-CD4 showed significant in vivo anti-tumor activity to inhibit thegrowth of human cancer cells in immuno-deficient mice (FIG. 5). Inaddition, TR-CD4 and tumor antigen-specific CD8⁺ T cells co-operativelyinhibited in vivo tumor growth (FIG. 5). Thus, the data presented hereinstrongly suggest that the recruitment of TR-CD4 at the local tumor sitepotentiate the anti-tumor immune responses, and accordingly will likelymake an effective and heretofore unavailable therapeutic approach forwidespread use in the clinic.

The following description provides illustrative examples of materialsand methods used to make and use various embodiments of the invention.

To develop a method to efficiently generate a large number of TR-CD4 bygene-engineering with tumor-recognizing T cell receptor (TCR) gene, fulllength TCR gene from three TR-CD4 clones were cloned and sequenced byusing 5′-RACE-PCR technique. The following TCRs were created:

-   -   1. HLA-DRB1*01-restricted NY-ESO-1:96-106-specific TR-CD4        (referred to herein as Clone: “SB95”)    -   2. HLA-DPB1*04-restricted NY-ESO-1:157-170-specific TR-CD4        (referred to herein as Clone: “5B8”)    -   3. HLA-DPB1*04-restricted NY-ESO-1:157-170-specific TR-CD4        (referred to herein as Clone: “JM”)

TCR genes from SB95 and JM were inserted into retroviral expressionvectors (such as MSCV-derived pMIG-II or pMIG-w vectors). A 5B8TCR-expressing vector is made in the same manner.

Retroviral transduction of these TCR genes efficiently transferredreactivity against cognate peptides to polyclonally expanded T cellsfrom peripheral blood mononuclear cells (PBMC) from healthy individuals.The nucleotide and amino acid sequences presented below represent thoseused to demonstrate the invention. The invention includes any and allpolynucleotide sequences encoding the amino acid sequences of the TCRconstructs described herein. Further, variations in amino acid sequencesin the TCRs are contemplated, so long as they do not adversely affectthe function of the TCR. In various embodiments, a TCR comprising one ormore amino acid changes as compared to the sequences presented hereinwill comprise conservative amino acid substitutions or othersubstitutions, additions or deletions, so long as the cells expressingthe recombinant TCRs of the invention can directly and specificallyrecognize tumor cells that express NY-ESO-1, wherein that recognition isdependent on expression of NY-ESO-1 and presentation of peptidesprocessed from it in an HLA class II restricted manner by the tumorcells. In embodiments, a TCR of the present invention comprises anyamino acid sequence that facilitates direct recognition of the tumorantigen on the tumor cells, without participation of an antigenpresenting cells. In embodiments, the amino acid sequence of a TCRprovided by this disclosure is at least 95%, 96%, 97%, 98% or 99%similar to an amino acid sequences provided in the sequence listing thatis part of this disclosure. In various embodiments, any TCR of theinvention can have a K_(off) value for its cognate epitope as definedherein that is essentially the same as the K_(off) for the cognateepitope exhibited by a TCR of a naturally occurring TR-CD4 for the sameepitope. In embodiments, the TCR amino acid sequences can comprisechanges in their constant region. In this regard, it is known in the artthat in general, the constant region of a TCR does not substantiallycontribute to antigen recognition. For example, it is possible toreplace a portion of the human constant region of a TCR with a murinesequence and retain function of the TCR. (See, for example, Goff S L etal. (2010) Cancer Immunology, Immunotherapy, 59: 1551-1560). Thus,various modifications to the TCR sequences disclosed herein arecontemplated, and can include but are not limited to changes thatimprove specific chain pairing, or facilitate stronger association withT cell signaling proteins of the CD3 complex, or inhibit formation ofdimers between the endogenous and introduced TCRs. In embodiments, theamino acid changes can be present in the CDR region, such as the CDR3region, including but not necessarily limited to substitutions of one,two, three, or more amino acids in the CDR3 sequence. In embodiments,the amino acid changes have no effect on the function of the TCR.

In specific and illustrative embodiments, the polynucleotide sequencesencoding the TCRs of the invention, and the amino acid sequences of theTCR α and TCR β chains encoded by the polynucleotides are as follows,wherein translation initiation and stop codons in the polynucleotidesequences are bold:

“JM” HLA-DPB1 *0401/0402-restricted NY-ESO-1₁₅₇₋₁₇₀-specific tumor-recognizing CD4⁺ T cell clone(a) cDNA nucleotide sequences of TCR α and β chains TCR α chain(SEQ ID NO: 1)

CACACAGCCAAATTCAATGGAGAGTAACGAAGAAGAGCCTGTTCACTTGCCTTGTAACCACTCCACAATCAGTGGAACTGATTACATACATTGGTATCGACAGCTTCCCTCCCAGGGTCCAGAGTACGTGATTCATGGTCTTACAAGCAATGTGAACAACAGAATGGCCTCTCTGGCAATCGCTGAAGACAGAAAGTCCAGTACCTTGATCCTGCACCGTGCTACCTTGAGAGATGCTGCTGTGTACTACTGCATCCCTAATAACAATGACATGCGCTTTGGAGCAGGGACCAGACTGACAGTAAAACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCT

TCR β chain (SEQ ID NO: 2)

TGGAGTCACTCAAACTCCAAGATATCTGATCAAAACGAGAGGACAGCAAGTGACACTGAGCTGCTCCCCTATCTCTGGGCATAGGAGTGTATCCTGGTACCAACAGACCCCAGGACAGGGCCTTCAGTTCCTCTTTGAATACTTCAGTGAGACACAGAGAAACAAAGGAAACTTCCCTGGTCGATTCTCAGGGCGCCAGTTCTCTAACTCTCGCTCTGAGATGAATGTGAGCACCTTGGAGCTGGGGGACTCGGCCCTTTATCTTTGCGCCAGCAGCTTCCCCAGGGAACCTAACTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAA

(b) amino acid sequences of TCR α and β chains (TCR variable  regions are in italic, CDR3 regions are in bold) TCR α chain(SEQ ID NO: 3) MKLVTSTTVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYI   50HWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDA  100

150 DFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANA  200FNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLK  250 VAGFNLLMTLRLWSS TCR β chain (SEQ ID NO: 4)MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRS   50VSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTL  100

150 EAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQP  200ALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV  250TQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALV  300 LMAMVKRKDF “5B8” HLA-DPB1 *0401/0402-restricted NY-ESO-1₁₅₇₋₁₇₀-specific tumor-recognizing CD4⁺ T cell clone(a) cDNA nucleotide sequences of TCR α and β chains TCR α chain(SEQ ID NO: 5)

CCCTGAGTTGCACATATGACACCAGTGAGAATAATTATTATTTGTTCTGGTACAAGCAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCAACGGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCTCAAGATCTCAGACTCACAGCTGGGGGACACTGCGATGTATTTCTGTGCTTTCTCGAGAGGGAGTGGAGGTAGCAACTATAAACTGACATTTGGAAAAGGAACTCTCTTAACCGTGAATCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCG

TCR β chain (SEQ ID NO: 6)

TGGAGTCTCCCAGTCCCCCAGTAACAAGGTCACAGAGAAGGGAAAGGATGTAGAGCTCAGGTGTGATCCAATTTCAGGTCATACTGCCCTTTACTGGTACCGACAGAGCCTGGGGCAGGGCCTGGAGTTTTTAATTTACTTCCAAGGCAACAGTGCACCAGACAAATCAGGGCTGCCCAGTGATCGCTTCTCTGCAGAGAGGACTGGGGGATCCGTCTCCACTCTGACGATCCAGCGCACACAGCAGGAGGACTCGGCCGTGTATCTCTGTGCCAGCAGCTTAGTCCCCGACAGTGCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAG

(b) amino acid sequences of TCR α and β chains (TCR variable  regions are in italic, CDR3 regions are in bold) TCR α chain(SEQ ID NO: 7) MAQTVTQSQPEMSVQEAETVTLSCTYDTSENNYYLFWYKQPPSRQMILVI  50

100

150 NVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSII  200PEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNL  250 LMTLRLWSS  TCR βchain (SEQ ID NO: 8) MGTRLLFWVAFCLLGADHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTA 50 LYWYRQSLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGGSVSTLTIQR  100

150 SEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQ  200PALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP  250VTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSAL  300 VLMAMVKRKDSRG “SB95” HLA-DRB1*0101-restricted NY-ESO-1₉₅₋₁₀₆-specific tumor-recognizing CD4⁺ T cell clone (a) cDNA nucleotide sequences of TCR αand β chains TCR alpha (SEQ ID NO: 9)

GTCGGTGACCCAGCTTGGCAGCCACGTCTCTGTCTCTGAGGGAGCCCTGGTTCTGCTGAGGTGCAACTACTCATCGTCTGTTCCACCATATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAGCTTCTCCTGAAGCACACAACAGGGGCCACCCTGGTTAAAGGCATCAACGGTTTTGAGGCTGAATTTAAGAAGAGTGAAACCTCCTTCCACCTGACGAAACCCTCAGCCCATATGAGCGACGCGGCTGAGTACTTCTGTGCTGTGAGTGATTCTAGGGCTGCAGGCAACAAGCTAACTTTTGGAGGAGGAACCAGGGTGCTAGTTAAACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCA

TCR beta (SEQ ID NO: 10)

GAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAATATGTTCTGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTACAGTGTCTCTAGAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCACCAACCAGACATCTATGTACCTCTGTGCCAGCAGATTCCCCGGGACAGCCTATAATTCACCCCTCCACTTTGGGAATGGGACCAGGCTCACTGTGACAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGA

(b) amino acid sequence of TCR α and β chains (TCR variable  regions are in italic, CDR3 regions are in bold) TCR α chain(SEQ ID NO: 11) MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVSVSEGALVLLRCNYSSSVPP  50YLFWYVQYPNQGLQLLLKHITGATLVKGINGFEAEFKKSETSFHLTKPSA  100

150 SSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWS  200NKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS  250VIGFRILLLKVAGFNLLMTLRLWSS  TCR β chain (SEQ ID NO: 12)MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHEN  50MFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESA  100

150 SEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQ  200PALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP  250VTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSAL  300 VLMAMVKRKDF 

Description of TCR expression vector. Viral transduction was performedusing a murine stem cell virus vector pMSCV-derived plasmid such aspMIG-II and pMIG-w (FIG. 6A). TCR-expressing constructs were insertedinto multiple cloning site (MCS) of pMIG plasmid. pMIG plasmids haveIRES-GFP after multiple cloning sites so that transduction efficacy ismonitored by GFP expression.

To induce equimolar expression of TCR α and β chain proteins, cDNAsencoding TCR α and β chain were connected by a linker sequence includingP2A translation skipping site (FIG. 6B(I)). Using this sequence, mRNA istranscribed as one sequence. Because of the ribosomal skipping by P2Asequence, two proteins were translated from the mRNA, to produceTCRβ-P2A fusion protein and P(Pro)-TCRα chain protein.

To avoid potential functional inhibition by P2A peptides added after theTCR β chain protein in TCR-expressing cassette (I), anotherTCR-expressing cassette that introduces the furin protease recognitionsite (RAKR) after TCR β chain gene was constructed (FIG. 6B(II). In thisexpression cassette, additional peptide after the TCR β chain protein isremoved by furin protease-mediated cleavage, resulting in expression ofTCR α and β chain proteins with minimal modification. In particular, inexpression cassettes with or without RAKR sequences, no amino acid isremoved relative to the sequences presented herein. However, for acassette without RAKR (FIG. 6B(I)), GSG linker and P2A sequences areattached to the C-terminus of beta chain, and a Proline (from P2A) isattached to the N-terminus of alpha chain. For a cassette with RAKR(FIG. 6B(II)), Arginine (from RAKR) is attached to the C-terminus of thebeta chain and Proline (from P2A) is attached to the N-teminus of alphachain. Thus, in embodiments, the expression vector encodes a fusionprotein comprising TCR amino acid sequences. In embodiments, the onlyTCR amino acid sequence is selected from the TCR amino acid sequencespresented herein.

The TCR-expressing sequences were cloned into multiple cloning site ofpMIG plasmid. Retrovirus was produced transiently or stably usingGP2-293 and PT67 packaging cell lines purchased from Clontech. Briefly,GP2-293 stably expresses viral gag-pol gene and they transiently produceafter co-transfection with pMIG and pVSV-G VSV-G viralenvelope-expressing plasmids. PT67 stably expresses viral gag-pol and10A1 viral envelope genes. After infection with retrovirus produced fromGP2-293, PT67 is integrated with the expression construct from pMIG, andtherefore stably (continuously) produces retrovirus. In an embodiment,promoter activity of 5′-LTR (long terminal repeat) is used to drivetransgene expression. However, other promoters such as EF-1α promotercan be introduced for enhancement of transgene expression.

Infection of retrovirus to PBMC-derived T cells. Whole PBMC wereobtained by a density gradient separation method and stored in a liquidnitrogen tank in 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide(DMSO) until use. PBMC (3−4×10⁶ cells/well in a 24-well culture plate)were polyclonally activated by 10 lag/ml phytohemaglutinin (PHA) for 2days in culture medium (RPMI1640 medium containing 10% FBS, L-Glutamine,Streptomycin, Penicillin and human recombinant IL-2). 1×10⁵ preactivatedPBMC in 100 μl culture medium were added to wells of a 96-well cultureplate pre-coated with 20-25 μg/ml retronectin in PBS and blocked with 2%bovine serum albumin (BSA) in PBS. In some experiments, 5 μg/ml anti-CD3monoclonal antibody (Clone:OKT3) was co-coated with retronectin. 100 μlsupernatant containing retrovirus was added to PBMC and incubated for 24hours. Retrovirus infection was performed 2-3 times every 24 hours.After infection, cells were expanded for 10-14 days and used forfunctional assays.

Results

High-titer retrovirus-producing PT67 clones were established. Thefollowing retrovirus-producing clones were established.

(1) pMIG-II/JM-TCR(II)

(2) pMIG-II/5 B 95 -TCR(II)

(3) pMIG-w/JM-TCR(I)

(4) pMIG-w/SB95-TCR(I)

(5) pMIG-w/JM-TCR(II)

(6) pMIG-w/SB95-TCR(II)

In the enumerated list above, (I) and (II) refer to expression cassetteswithout and with the furin protease recognition site (RAKR),respectively, as shown in FIG. 6B. The transduction efficacy measured byGFP expression after a single infection to Jurkat cells was: 60% for(1); 55% for (2); 75% for (3); 75% for (4); 64% for (5); and 62% for(6).

Retrovirus vectors (1) and (2) were transduced to polyclonally activatedPBMC. Transduction efficacy as measured by GFP expression was about40-50%. The reactivity of retrovirally expressed TCR was tested againstthe same NY-ESO-1-derived cognate peptides (NY-ESO-1:91-110 for SB95-TCRand NY-ESO-1:157-170 for JM-TCR) that were recognized by the originalTR-CD4 clones. Significantly more IFN-γ was produced againstpeptide-pulsed target cells than peptide-unpulsed target cells (FIG. 7),which demonstrates that the cloned TCR genes are functional to transferthe same antigen specificity of original TR-CD4 clones when they aretransduced by viral vectors. Functional testing of the remaining TCRexpression vectors can be performed in the same manner, such as byinfecting activated human peripheral blood mononuclear cells withretrovirus carrying any TCR gene disclosed herein. TCR gene-transducedand untransduced cells can be cocultured for 24 hours withNY-ESO-1-expressing cell lines or tumor samples, and IFN-γ produced bythe transduced cells determined using any suitable means, such as byELISA. IFN-γ level in the supernatant by TCR gene-transduced cells willbe higher when co-cultured with cells that express NY-ESO-1 or NY-ESO-1peptide-pulsed cells, whereas cells cocultured with cells that do notexpress NY-ESO-1 will have significantly less IFN-γ production.Likewise, negative controls, such as untransduced cells, will havesignificantly less IFN-γ production. Thus, transfection with arepresentative recombinant TCR will result in the capability of thecells into which a polynucleotide encoding the TCR to have the sameantigen-specificity which directly recognizes NY-ESO-1 antigen on cancercells.

Although the invention has been described in detail for the purposes ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

We claim:
 1. A modified human T cell comprising a recombinantpolynucleotide encoding a T cell receptor (TCR), wherein the T cell iscapable of direct recognition of a cancer cell expressing a NY-ESO-1antigen, wherein the direct recognition of the cancer cell compriseshuman leukocyte antigen (HLA) class II-restricted binding of the TCR tothe NY-ESO-1 antigen expressed by the cancer cell.
 2. The modified cellof T cell claim 1, wherein the recombinant polynucleotide encodes a TCRalpha chain having the sequence of SEQ ID NO:3 and a TCR beta chainhaving the sequence of SEQ ID NO:4, or a TCR alpha chain having thesequence of SEQ ID NO:7 and a TCR beta chain having the sequence of SEQID NO:8, or a TCR alpha chain having the sequence of SEQ ID NO:11 and aTCR beta chain having the sequence of SEQ ID NO:12.
 3. The modified cellof claim 1, wherein the sequence encoding the alpha chain and/or thebeta chain does not comprise introns.
 4. A method for prophylaxis and/ortherapy of an individual diagnosed with, suspected of having or at riskfor developing or recurrence of a cancer, wherein the cancer comprisescancer cells which express NY-ESO-1 antigen, the method comprisingadministering to the individual modified human T cells comprising arecombinant polynucleotide encoding a T cell receptor (TCR), wherein theT cells are capable of direct recognition of the cancer cells expressingthe NY-ESO-1 antigen, wherein the direct recognition of the cancer cellscomprises human leukocyte antigen (HLA) class II-restricted binding ofthe TCR to the NY-ESO-1 antigen expressed by the cancer cells.
 5. Themethod of claim 4, wherein the modified human T cells are CD4⁺ T cells.6. The method of claim 4, wherein the recombinant polynucleotide encodesa TCR alpha chain having the sequence of SEQ ID NO:3 and a TCR betachain having the sequence of SEQ ID NO:4, or a TCR alpha chain havingthe sequence of SEQ ID NO:7 and a TCR beta chain having the sequence ofSEQ ID NO:8, or a TCR alpha chain having the sequence of SEQ ID NO:11and a TCR beta chain having the sequence of SEQ ID NO:12.
 7. The methodof claim 5, wherein the cancer cells are selected from bladder cancercells, brain cancer cells, breast cancer cells, gastric cancer cells,esophageal cancer cells, head and neck cancer cells, hepatobiliarycancer cells, kidney cancer cells, ovary cancer cells, non-small celllung cancer cells, myeloma, prostate cancer cells, sarcoma cells,testicular cancer cells, melanoma cells, or combinations thereof.
 8. Themethod of claim 5, comprising removing CD4+ T cells from the individualprior to the administering, and modifying the CD4+T cells by introducinginto the CD4+ T cells the recombinant polynucleotide encoding the TCR.9. An expression vector encoding a T cell receptor (TCR), wherein theTCR comprises an alpha chain having the sequence of SEQ ID NO:3 and abeta chain having the sequence of SEQ ID NO:4, or an alpha chain havingthe sequence of SEQ ID NO:7 and a beta chain having the sequence of SEQID NO:8, or an alpha chain having the sequence of SEQ ID NO:11 and abeta chain having the sequence of SEQ ID NO:12.
 10. A method for makingan expression vector encoding a recombinant T cell receptor (TCR), themethod comprising obtaining a plurality of T cells from an individual,identifying T cells that are capable of direct recognition of a cancercell expressing a NY-ESO-1 antigen in an HLA class II-restricted mannerwithout antigen presenting cells presenting the NY-ESO-1 antigen to theT cells, determining the sequence of the alpha chain of the TCR and thesequence of the beta chain of the TCR, and introducing into anexpression vector a polynucleotide sequence encoding the alpha chain ofthe TCR and the beta chain of the TCR.
 11. The method of claim 10,further comprising introducing the expression vector into a cell suchthat the TCR is expressed.